Jacqueline Sagen - US grants

During the past several years, work in our laboratory has indicated that the transplantation of adrenal medullary chromaffin cells into CNS pain modulatory regions is a potentially complementary and novel approach in the therapeutic management of pain. Neural transplants may be a means to achieve sustained delivery of pain-reducing neuroactive substances to CNS pain modulatory regions on a long-term basis, reducing or eliminating the need for repeated narcotic administration. Since the encouraging results of these studies has brought this work close to entering clinical trials as a therapy for pain reduction, it is essential to establish optimal conditions for graft selection and handling. The goals of the current proposal are to further characterize the transplants behaviorally, pharmacologically, biochemically, and histologically, to determine optimal graft sources for long term survival and continued production of pain- reducing neuroactive substances, and to develop cell lines for transplantation which can provide a uniform and readily available source of opioid peptides and other agents for the long-term alleviation of chronic pain. Pain reduction over long periods of time will be assessed using acute and chronic pain tests. Biochemical assays and pharmacologic studies will be done to explore the mechanisms of action of the transplants, interactions with other agents, and responses to environmental stimuli. The issues of tolerance and tachyphylaxis will also be addressed. It is possible that the combined release of subeffective levels of pain-reducing neuroactive substances from the transplants can synergize to produce potent analgesia without the development of significant tolerance. Since adrenal medullary allografts are the most likely donor source in the initial clinical phases, it is important to determine the optimal conditions for allograft survival. Short term maintenance in explant culture may improve the survival and transplantability of adrenal medullary allografts. However, the limited availability and non-uniformity of human adrenal medullary tissue reduces its feasibility for widespread use. Thus, another aim of the proposed work is to explore alternative donor sources of chromaffin cells for transplantation. A potentially useful source of chromaffin cells is the bovine adrenal gland since large quantities are readily available. However, the safety and efficacy of such xenografts must be established. One way to immunologically isolate bovine chromaffin cells is to encapsulate them in transplantable permselective membranes. In addition, encapsulation will limit migration of transplanted cells from critical CNS placement sites. Finally, a long term goal of the proposed studies is to develop defined and uniform cells lines for transplantation which are engineered to produce high levels of opioid peptides.

Disturbances of mood are among the most common and most debilitating of psychiatric disorders. A large proportion of patients with major depressive disorders suffer chronic or recurrent symptoms. The cost of depression is great, both in human and economic terms. While currently available antidepressant therapies are effective in many cases, they are limited by potentially serious side effects, poor patient compliance, and intentional overdose. Clinical depression most likely involves a disturbed and imbalanced functioning of CNS monoaminergic systems. A novel approach towards restoring imbalanced functioning in the CNS is the use of neural transplantation. When placed in appropriate regions, neural grafts can serve as a long-term and readily available source of neuroactive substances. A significant advantage of this approach for the chronically depressed patient would be the ability to provide a continually renewable source of monoamines on a long-term or permanent basis, reducing or eliminating the need for repeated antidepressant administration. The proposed study is an initial attempt to assess the potential for neural transplants to alleviate depression. To assess the potential for transplants of monoamine-producing cells to alleviate depression, two well established animals models will be used initially; the learned helplessness model and the behavioral despair model. The learned helplessness model is the best studied and is supported by a number of parallels between clinical depression and laboratory observations, including weight loss, lethargy, and reduced motivation, as well as similarities in biochemistry and treatment. The behavioral despair model is particularly sensitive as a screen for clinical antidepressants. Monoaminergic graft sources will include the pineal gland, which produces high levels of serotonin, and the adrenal medulla, which produces high levels of norepinephrine. Solid tissue, explants, and cell suspensions will be tested for ability to reduce behavioral deficits in the ra t depression models. Initially the transplants will be placed in the frontal neocortex, since this site has been implicated in human depression, as well as in animal depression models. The long term potential for neural transplants to reduce behavioral depression will be assessed over time, ranging from 1 week to 1 year. Specific antagonists will be used to assess the contribution of substances released from the grafts to the reduction of behavioral depression. The ability for the neural transplants to reduce deficits following neurotoxin administration will also be tested. Graft viability and function will be determined over time using biochemical assays and immunocytochemistry.

DESCRIPTION: (Applicant's abstract) Studies in this laboratory and elsewhere have shown that transplants of adrenal chromaffin cells into the spinal subarachnoid space can result in reduction in "pain" behaviors in rat models of persistent pain. In a continuation of this work, experiments are proposed using rats with persistent pain due to experimentally-induced arthritis or sciatic nerve constriction. Two primary goals will be addressed; these are: 1) to assess the safety of adrenal medullary and chromaffin cell transplants in the spinal subarachnoid space; and 2) to clarify the mechanisms of reduction of pain behaviors consequent to adrenal medullary transplants, with particular attention to analysis of the effects on neuroplastic remodeling and long-term neuropathological consequences of chronic pain. The "safety" issue will be addressed by analysis of three factors: 1) motor and other physiological functions; 2) cerebrospinal fluid and blood serum, and 3) neuropathology in spinal cord tissues. The effects of adrenal medullary transplants on functional plasticity in the central nervous system will be examined in the animal pain models with a combination of behavioral measurements and measurements of immediate early gene expression in spinal and supraspinal structures. Evaluation of the benefits of transplants on long-term pathological processes will be examined histologically by analysis of spinal tissues for the presence of hyperchromatic ("dark") neurons and for changes in the numbers of neurons expressing the inhibitory neurotransmitter GABA.

DESCRIPTION (provided by applicant): Neurogenic pain resulting from injury to the nervous system is often persistent and debilitating, and presents a significant clinical challenge as it does not respond well to traditional therapies. Novel interventive strategies using cellular transplantation into the spinal cord have been explored to provide a local and continually renewable source of analgesic agents and neurotrophic factors or to replace lost cellular populations. However, the selection of appropriate cells for transplantation is challenging, due to limitations in human organ donor availability, immunogenicity, and potential disease transmission. The goal of this R21 Exploratory grant proposal is to evaluate the feasibility of using autologous stem cell-like bone marrow cells as vehicles for delivering analgesic agents to local spinal sites for alleviation of neurogenic pain. The use of autologous cells that can be expanded ex vivo would overcome the limitations associated with other donor sources, as well as the potential ethical objections surrounding the use of embryonic stem cells. In addition, bone marrow stromal cells have been shown to migrate selectively and "home" to damaged regions of the CMS when injected intravenously, and thus may offer a minimally invasive grafting approach. In order to evaluate this approach for pain due to peripheral or central injury, a unique subpopulation of human bone marrow stromal cells which have been shown to exhibit embryonic stem cell self-renewal and differentiation properties (named the Marrow-Isolated Adult Multilineage Inducible or MIAMI cells), will be used. Cells will be administered intravenously or intrathecally and assessed for their ability to target the spinal cord and to deliver analgesic agents using two distinct models of neurogenic pain: the quisqualic acid (QUIS) model for excitotoxic spinal cord injury pain and the chronic constriction injury (CCI) model for peripheral neuropathic pain. The first produces a fairly large lesion site associated with overt neuronal loss in the dorsal horn, while the second produces a more subtle pathology and reorganization in the dorsal horn, both resulting in abnormal pain processing including allodynia, hyperalgesia, and dysesthesias. Two specific aims are proposed; the first to evaluate the ability of the MIAMI cells to home to sites of pathology in the dorsal horn following QUIS or CCI, and the second to evaluate potential cell-mediated delivery of analgesic agents to these sites using cells engineered to produce proenkephalin as a proof of concept. Evaluations will include alterations in pain behaviors, host-graft integration, spinal neurochemistry, and potential pathology. If successful, findings from these studies should lead to development of novel and potent strategies in the management of chronic neurogenic pain.

[unreadable] DESCRIPTION (provided by applicant): Pain from injury to the peripheral or central nervous system is often persistent and debilitating, and presents a significant clinical challenge as it does not respond well to traditional therapies. The underlying hypothesis and motivation for the proposed studies is that severe chronic pain from spinal cord or peripheral nerve injury results from loss of spinal inhibitory processes and consequent abnormal hyperexcitability in dorsal horn pain transmission neurons, and that restoration of spinal inhibition by neural transplantation will alleviate neuropathic pain. In order to accomplish this, peripheral and central models of injury-induced pain will be evaluated for neuropathology and stem cell transplantation strategies. The chronic constriction injury model (CCI) will be used for peripheral neuropathic pain and the quisqualic acid model (QUIS) for excitotoxic spinal cord injury pain. Aim 1 will characterize and compare the inhibitory neuronal cell loss in the spinal dorsal horn and consequent exaggerated pain following CCI and QUIS. Aim 2 will characterize and compare the abnormal activation and hyperexcitability of spinal dorsal horn neurons following CCI and QUIS. Studies in these 2 aims will include in depth evaluation of morphological and neurochemical changes in the spinal dorsal horn that likely contribute to inhibitory loss and abnormal hyperexcitability (GABA neuronal loss or dysfunction), alterations in sensory processing (exaggerated behavioral responses and c-fos activation in response to noxious and innocuous stimuli), and physiological alterations in dorsal horn neuronal excitability to establish a comparative basis between the models and treatment interventions. Aims 3-5 will explore the use of neural progenitor transplants to replace lost or dysfunctional inhibitory neurocircuitry in the spinal dorsal horn following peripheral nerve or spinal cord injury. Aim 3 will generate a reliable and reproducible source of GABAergic neural progenitor cells for transplantation using extrinsic manipulation (trophic factor shock), genetic manipulation (blocking HLH transcription factor Hes1 to promote GABAergic differentiation) and/or cell selection (GAD promoter-GFP and FACS sorting). Aim 4 will evaluate GABAergic neural progenitor transplantation strategies in restoring spinal inhibition and alleviating chronic neuropathic and central pain. Aim 5 will evaluate chromaffin cell and GABAergic neural progenitor co-grafting strategies, as chromaffin cells produce a cocktail of trophic factors which improve neural stem cell survival and differentiation, and can reduce chronic neuropathic and SCI pain. Findings from these studies should lead to improved interventive strategies in the management of intractable neuropathic pain. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Chronic pain is a frequently debilitating and poorly understood consequence of spinal cord injury (SCI). In addition to reduced quality of life, the presence of untreated pain can interfere with the ability to fully participate in rehabilitative strategies, thus reducing potential long-term gains in functional recovery. SCI pain is notoriously difficult to treat, and the complexities of available animal models impede the rapid identification and screening of promising pharmacotherapies and novel interventive strategies. The goal of this exploratory/developmental R21 proposal in response to PA-06-542 (Mechanisms, models, measurement, & management in pain research) is to develop a strong predictive model to streamline this process and facilitate translation of promising therapies more rapidly to the clinic. Our laboratory is exploring a spinal compression injury model which can overcome the complications of other SCI pain models, particularly for below-injury neuropathic pain. Preliminary findings demonstrate robust and persistent reproducible SCI pain symptoms. Thus, the spinal compression model offers an opportunity to generate a rapid screening formula for evaluating the effects of clinically relevant analgesic drugs and novel therapeutics. The primary objectives will be: 1) To characterize the compression injury model for use in rapid screening for SCI pain, using a battery of evoked and spontaneous outcome measures; 2) To develop a model incorporating these behavioral outcomes using agents that are already approved and readily available for clinical use; 3) To utilize this rapid screening model for evaluation of potentially synergistic combination strategies and serve as guidance for achieving substantial improvement in the management of chronic SCI pain. Initially, clinically approved pharmacologic agents will be evaluated, as they are likely to have untapped potential efficacy in reducing SCI pain, particularly when administered in selected and synergistic combinations and, since they face fewer regulatory hurdles, can be more rapidly brought to patients. Since patients experience a constellation of symptoms, evaluation will include tests for mechanical allodynia, heat hyperalgesia, cold hypersensitivity, and ongoing spontaneous pain (Aim 1). A model will be developed for selection of promising agents, based initially on efficacy in reducing these various neuropathic pain symptoms, and incrementally adjusted as data is collected (Aim 2). Potential novel synergistic combinations will be done using isobolographic analysis (Aim 3). The co-administration of agents with distinct mechanisms should allow them to be given in substantially lower doses with reduced untoward side effects, and can result in potent analgesia with subeffective or marginally effective doses of individual agents. If successful, the development of this approach should rapidly accelerate the process of bringing effective analgesic therapies, including novel interventive strategies, to SCI patients with chronic pain. [unreadable] [unreadable] Project Narrative: Following spinal cord injury, many patients suffer from long-lasting pain, which can be severe and debilitating, and limit participation in rehabilitation programs, resulting in poorer prognosis and reduced functional recovery. Pharmacological options for patients with SCI pain are limited and marginally effective in current practice, and thus SCI pain is notoriously difficult to treat. The goal of the proposed studies is to develop and implement a predictive and efficient model for rapidly screening and translating promising analgesic therapies to markedly improve the treatment of clinical spinal cord injury pain. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chronic neuropathic pain following spinal cord injury (SCI) is a particularly challenging clinical target, as traditional analgesic drugs are only marginally effective at best, and fraught with untoward side effects owing to the high doses required. In addition to direct interference in the quality of life of these patients, the presence of untreated pain can limit participation in rehabilitation programs, resulting in poorer long-term prognosis and reduced functional recovery. Our laboratory has been evaluating traditional and non-traditional analgesics alone and in combinations for their ability to reduce neuropathic pain symptoms in a spinal clip compression model for SCI pain. This model produces persistent SCI pain symptoms, and demonstrates the ability to successfully distinguish pharmacologic agents with modest clinical efficacy from those reported to be ineffective in reducing clinical SCI pain. Marine cone snail peptides (conopeptides) have emerged as particularly promising candidates for this indication. Cone snails produce a number of novel peptides with selective ion channel blocking activity, and have become a major focus for new drug development in the treatment of CNS injury and disease. Among those potentially targeting pain pathways are the omega- conopeptide MVIIA, a peptide with N-type Ca2+ channel blocking activity (which has reached FDA approval as derivative Prialt in the treatment of severe pain) and conantokin-G (conG), a peptide with NMDA antagonist activity. Recent findings in our lab showed robust dose-dependent amelioration of pain responses following bolus intrathecal injection of these conopeptides, without apparent untoward side effects, and a highly potent synergistic pain reduction when used together in combination. These findings are remarkable in light of the difficulty in obtaining even modest effects of other drug combinations in this model or in clinical SCI pain reports. The goal of this exploratory/developmental R21 proposal in response to PA-10-007 (Mechanisms, models, measurement, &management in pain research) is to 1) explore the potential for long-term intrathecal delivery of conopeptide combinations to alleviate chronic SCI pain, and 2) to develop a means for providing sustained conopeptide delivery using gene therapy or transplanted cells. Specific aims to accomplish this are 1) To characterize neuropathic pain-reducing ability and effective dosing, time-course, and side effects of chronic conopeptide administration;2) To evaluate the potential for long-term delivery of conopeptides via gene therapy or cell-based strategies. Initial studies will focus on adult bone marrow stem cells, which can be autologously derived and are currently being developed in clinical trials for SCI and other indications. Adeno- associated virus (AAV)-mediated delivery will also be explored in these initial studies, owing to its reported safety and use in clinical trials for other neurological disorders. If successful, this project should accelerate the process of bringing effective analgesic therapies to spinal cord injured patients, and advance novel high-impact strategies in the therapeutic management of debilitating chronic pain. PUBLIC HEALTH RELEVANCE: Chronic pain following spinal cord injury is estimated to occur in up to 70% of patients, with at least one-third rating it as so severe that it is their primary impediment to participation in daily activities and social well-being. Neuropathic pain following SCI is particularly resistant to treatment, and its persistence can limit participation in rehabilitation therapies and further reduce functional recovery. Thus there is a critical need for identifying effective long-term therapies for alleviating chronic SCI pain. The proposed studies will evaluate the potential for novel and remarkably selective naturally-derived peptides (conopeptides), delivered on a long-term basis via gene therapy or cell transplantation approaches, to ameliorate chronic pain following spinal cord injury.